Methods for treating patients at risk for costimulation blockade resistant rejection with belatacept

ABSTRACT

The present invention provides methods utilizing changes in CD4+CD57+ T cells levels for determining the susceptibility of a transplant patient or patient in need thereof to costimulation blockade resistant rejection. These methods are useful for identifying effective drug regimens for the treatment of immune disorders associated with graft transplantation and/or maintenance of a transplant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/779,204, filed Sep. 22, 2015, now abandoned, which is the 371 ofInternational Application No. PCT/US2014/31945, filed Mar. 27, 2014, nowpublished, which claims priority to U.S. Provisional Application No.61/806,206, filed Mar. 28, 2013; the entire content of which isincorporated herein by reference.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

This invention relates to methods for determining the susceptibility ofa transplant patient to costimulation blockade resistant rejection.These methods are useful for identifying effective drug regimens for thetreatment of immune disorders associated with graft transplantationand/or maintenance of a transplant.

SEQUENCE LISTING

Applicants enclose herewith the “SEQUENCE LISTING” for theabove-captioned application in ASCII format via EFS-Web and is herebyincorporated by reference in its entirety. The ASCII copy, created onSep. 22, 2015, is named SEQ.txt and is 6 kilobytes in size. Theinformation contained in the attached “SEQUENCE LISTING” is identical tothe information in the specification as originally filed. No new matteris added.

BACKGROUND OF THE INVENTION

Given the central role of T-cells in transplant rejection, a common goalamong current immunosuppressive therapies is to block T-cell activationand function (Sayegh, M. H. et al., N. Engl. J. Med., 338(25):1813-1821(1998)). T-cells require both an antigen-specific (Signal 1) andco-stimulatory signal (Signal 2) for full activation (Lenschow, D. J. etal., Annu. Rev. Immunol., 14:233-258 (1996)). One of thebest-characterized co-stimulatory pathways involves the CD28-CD80/86(B7-1/2) interaction (Linsley, P. S. et al., Annu. Rev. Immunol.,11:191-212 (1993)). Cytotoxic T-lymphocyte antigen 4 (CTLA4) binds toCD80/86 with higher avidity than CD28, and is transiently expressed onT-cells following their activation, where it interrupts the interactionbetween CD28 and CD80/86 (Oosterwegel, M. A. et al., Curr. Opin.Immunol., 11(3):294-300 (1999)). This creates a negative feedback signalfor T-cell activation.

Functionally distinct T cell populations can be defined by theexpression of specific cell surface antigens. Numerous studies describeassociations between T cell surface phenotype and function (Appay, V. etal., Nat. Med., 8:379-385 (2002)). CD57, a surface molecule expressed onT and NK cells, is a marker with functional associations. CD57 has beencommonly described as an external marker of cell senescence, a state inwhich the cell is in persistent cell cycle arrest. Expansions of CD4+ Tcells expressing CD57 have been associated with a number of chronicpathological conditions such as tuberculosis, malaria, rheumatoidarthritis, and HIV-1 infection (Maeda, T. et al., Arthritis Rheum.,46:379-384 (2002); Imberti, L. et al., Blood, 89:2822-2832 (1997)).

Belatacept (L104EA29YIg) is the first biologic agent approved forprimary maintenance immunosuppression, selectively blocking the CD28co-stimulation pathway to prevent T-cell activation (Larsen, C. P. etal., Am. J. Transplant., 5:443-453 (2005)). Belatacept combined withcorticosteroids and a mycophenolic acid is indicated for prophylaxis ofgraft rejection in adults receiving a renal transplant. While Belatacepthas been associated with improved long-term graft function as well aspatient and graft survival, patients treated with this costimulationblockade molecule tended to experience early acute rejection.

Further, while the introduction of kidneys from extended criteria donorshas been necessary to alleviate donor organ shortages, recipients are atgreater risk of complications and graft loss than recipients of livingor standard criteria deceased donor kidneys (Tullius, S. G. et al., Eur.Renal Dis., 1:51-54 (2007)).

With the increasing use of costimulation blockade molecules and higherrisk extended criteria donor organs, it is clear that there is a need toprospectively identify patients at risk for costimulation blockaderesistant rejection. The Inventors have identified a CD4+ T cellphenotype that strongly correlates with risk for costimulation blockaderesistant rejection.

At present, there are no predictive assays to identify patients at riskof costimulation blockade resistant rejection. Thus, there is a need inthe art for a diagnostic assay that could determine whether a patientwould be eligible to be treated with costimulation blockade molecules orat risk of acute rejection. The present invention provides such novelassay.

SUMMARY OF INVENTION

In one aspect, the invention provides a method of predicting if atransplant patent is at risk of costimulation blockade resistantrejection, the method comprising: (a) acquiring a blood sample from aperson receiving costimulation blockade therapy, (b) quantitating thepercent of CD+CD57+ T cells and (c) comparing the percentage ofCD4+CD57+ T cells in the test sample with the percentage of CD4+CD57+ Tcells in a reference, wherein the percentage of CD4+CD57+ T cells in thetest sample is elevated compared to a reference.

In another aspect, the invention provides a method of predicting if apatient in need of a transplant is at risk of costimulation blockaderesistant rejection, the method comprising: (a) acquiring a peripheralblood sample from a person prior to transplantation, (b) quantitatingthe percent of CD+CD57+ T cells and (c) comparing the percentage ofCD4+CD57+ T cells in the test sample with the percentage of CD4+CD57+ Tcells in a reference, wherein the percentage of CD4+CD57+ T cells in thetest sample is elevated compared to a reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a graph of PBMCs from belatacept treated patients examinedfor markers of senescence (CD57) and exhaustion (PD1). Patients whoexperienced ACR had a significantly higher percentage of CD57+PD1− cellsin the CD4+, but not CD8+, compartment. These differences weresignificant in each memory subset of CD4+, but not CD8+, T cells. TCMdenotes Central Memory T cells; TEM denotes Effector Memory T cells;TEMRA denotes Terminally Differentiated Effector Memory T cells.

FIG. 2 shows a graph of PBMCs from tacrolimus treated patients examinedfor markers of senescence (CD57) and exhaustion (PD1). No significantcorrelations with outcome were found in the CD57+PD1- compartment ofeither CD4+or CD8+T cells. TCM denotes Central Memory T cells; TEMdenotes Effector Memory T cells; TEMRA denotes Terminally DifferentiatedEffector Memory T cells.

FIG. 3 shows that CD28− T cells do not predict acute cellular rejectionoutcome. PBMCs from belatacept and tacrolimus treated patients wereexamined for surface expression of costimulation markers CD28 and CD27.A trend existed towards increased prevalence of CD28 negative CD4 Tcells in the belatreated patients who rejected, but this did not reachstatistical significance. No correlations between CD28 expression on CD8cells and outcome were observed.

FIG. 4 shows the phenoflow panels utilized in Example 1. *CD3 antibodywas diluted 1:8 before use, while the CD45RA antibody was diluted 1:10before use.

FIG. 5 (SEQ ID NOS: 1 and 2) depicts a nucleotide and amino acidsequence of L104EA29YIg, a CTLA4 mutant molecule comprising a signalpeptide; a mutated extracellular domain of CTLA4 starting at methionineat position +1 and ending at aspartic acid at position +124, or startingat alanine at position −1 and ending at aspartic acid at position +124;and an Ig region. SEQ ID NOS: 1 and 2 depict a nucleotide and amino acidsequence, respectively, of L104EA29YIg, a CTLA4 mutant molecule,comprising a signal peptide; a mutated extracellular domain of CTLA4starting at methionine at position +27 and ending at aspartic acid atposition +150, or starting at alanine at position +26 and ending ataspartic acid at position +150; and an Ig region.

DETAILED DESCRIPTION OF THE INVENTION Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, “ligand” refers to a molecule that specificallyrecognizes and binds another molecule, for example, a ligand for CTLA4is a B7 molecule.

“L104EA29YIg”, “belatacept” and “LEA29YIg” are used interchangeableherein and refer to a fusion protein that is a soluble CTLA4 mutantmolecule comprising an extracellular domain of wildtype CTLA4 with aminoacid changes A29Y (a tyrosine amino acid residue substituting for analanine at position 29) and L104E (a glutamic acid amino acid residuesubstituting for a leucine at position +104), joined to an Ig tail(included in FIG. 4, SEQ ID NOS: 1 and 2; DNA encoding L104EA29YIg wasdeposited on Jun. 20, 2000 with ATCC® number PTA-2104. Belatacept iscommercially available as the IV formulation, NULOJIX® (Bristol-MyersSquibb Company). Belatacept is a fusion protein that acts as a selectivecostimulation blocker by binding surface ligands (CD80 and CD86) onantigen-presenting cells. In antigen recognition, the binding of thecostimulation receptor on T cells (CD28) to CD80 and CD86 (signal 2) isrequired for efficient T-cell activation when the T-cell receptorencounters MHC alloantigens (signal 1).

As used herein, “B7” refers to the B7 family of molecules including, butnot limited to, B7-1 (CD80), B7-2 (CD86) and B7-3 that may recognize andbind CTLA4 and/or CD28.

As used herein, the term “CD4” refers to “cluster of differentiation 4”which is a glycoprotein expressed on the surface of T helper cells,monocytes, macrophages, and dendritic cells

As used herein, “CD57+ T cells” are T cells with CD57 moleculesexpressed on the cell surface.

As used herein, to “block” or “inhibit” a receptor, signal or moleculemeans to interfere with the activation of the receptor, signal ormolecule, as detected by an art-recognized test. Blockage or inhibitionmay be partial or total. For example, blockage of a cell-mediated immuneresponse can be detected by determining the functionality of thetransplant, such as the serum creatinine concentrations following renaltransplantation.

As used herein, “costimulation blockade resistant rejection” and “acuterejection” are used interchangeable herein and refer to a reactionagainst allograft or xenograft tissue that is incompatible with therecipient, which typically occurs 2 to 60 days after transplantation.

As used herein, “treat” or “treating” a disorder or disease means tomanage a disease or disorder by medicinal or other therapies. Treatmentof a disease or disorder may suppress immune-mediated events associatedwith a disease, ameliorate the symptoms of a disease or disorder, reducethe severity of a disease or disorder, alter the course of disease ordisorder progression and/or ameliorate or cure the basic disease ordisorder problem. For example, to treat an immune disorder associatedwith graft transplantation may be accomplished by regulating an immuneresponse e.g., by regulating functional CTLA4- and/or CD28-positive cellinteractions with B7-positive cells. For example, treating renaltransplant rejection includes inhibition of renal transplant rejectionas measured by glomerular filtration rate (GFR). For example treatingimmune disorders associated with graft transplantation includesprophylaxis of organ rejection by administration of L104EA29YIg.Further, treating immune disorders associated with graft transplantationmay prolong the survival of the host and transplanted organ.

As used herein, “immune disorders associated with graft transplantation”means any transplant related disease mediated by T-cell interactionswith B7-positive cells including, but not limited to, immune disordersassociated with graft transplantation rejection, graft relateddisorders, graft versus host disease (GVHD) (e.g., such as may resultfrom bone marrow transplantation, or in the induction of tolerance),rejection of the graft or transplant including acute rejection of thegraft or transplant and chronic rejection of the graft or transplant.

As used herein, the “graft” or “transplant” may be solid organallografts or xenografts, tissue or cell allografts or xenografts orexternal anatomy allografts or xenografts, including but not limited toskin, islet cells (also known as islets), muscles, hepatocytes, neurons,heart, liver, kidney, lung, appendages, limbs, nose, ear or face.

Early costimulation blockade resistant rejection has been seen in somepatients on costimulation blockade immunosuppression, such asbelatacept. Hypothesizing that T cell phenotype might prospectivelyidentify patients at risk for costimulation blockade resistantrejection, transplant patients receiving costimulation blockade, werestudied by specifically quantifying terminally differentiated memorycells expressing CD57, a marker of T cell senescence, and relating thisexpression to clinical outcome (see Example 1).

As discussed in Example 1, peripheral blood samples were obtained fromkidney transplant patients prior to drug administration and analyzed bymultiparameter flow cytometry, specifically analyzing the samples formarkers of memory, differentiation, activation, exhaustion andsenescence. Patients who eventually experienced acute rejection had amarkedly higher percentage of CD4+CD57+PD1− T cells (p=0.0055) at thetime of transplantation compared to patients without rejection (FIG. 1).Although typically considered a marker of senescence, CD57 expression onCD4+ T cells was paradoxically correlated with expression of theproliferation marker Ki67 (p=0.0087), and was not associated withshorter telomere length (p=0.7424). These relationships were limited tothe CD4 compartment. CD8+CD57+ T cells marginally trended higher inacute rejection patients (p=0.2358, FIG. 1), but CD57 expression on CD8+T cells was associated with more traditional indices of senescenceincluding reduced Ki67 expression and shorter telomere length.

In one aspect, the invention provides a method of predicting if atransplant patent is at risk of costimulation blockade resistantrejection, the method comprising: (a) acquiring a blood sample from aperson receiving costimulation blockade therapy, (b) quantitating thepercent of CD+CD57+ T cells and (c) comparing the percentage ofCD4+CD57+ T cells in the test sample with the percentage of CD4+CD57+ Tcells in a reference, wherein the percentage of CD4+CD57+ T cells in thetest sample is elevated compared to a reference.

In another aspect, the invention provides a method of predicting if apatient in need of a transplant is at risk of costimulation blockaderesistant rejection, the method comprising: (a) acquiring a peripheralblood sample from a person prior to transplantation, (b) quantitatingthe percent of CD+CD57+ T cells and (c) comparing the percentage ofCD4+CD57+ T cells in the test sample with the percentage of CD4+CD57+ Tcells in a reference, wherein the percentage of CD4+CD57+ T cells in thetest sample is elevated compared to a reference.

In one embodiment, the invention provides a method of predicting if akidney transplant patent is at risk of costimulation blockade resistantrejection, the method comprising: (a) acquiring a blood sample from aperson receiving costimulation blockade therapy, (b) quantitating thepercent of CD+CD57+ T cells and (c) comparing the percentage ofCD4+CD57+ T cells in the test sample with the percentage of CD4+CD57+ Tcells in a reference, wherein the percentage of CD4+CD57+ T cells in thetest sample is elevated compared to a reference.

In one embodiment, the invention provides a method of predicting if apatient in need of a kidney transplant is at risk of costimulationblockade resistant rejection, the method comprising: (a) acquiring aperipheral blood sample from a person prior to transplantation, (b)quantitating the percent of CD+CD57+ T cells and (c) comparing thepercentage of CD4+CD57+ T cells in the test sample with the percentageof CD4+CD57+ T cells in a reference, wherein the percentage of CD4+CD57+T cells in the test sample is elevated compared to a reference.

In one embodiment, the reference may be blood from normal healthycontrols or stable transplant patients; or a reference value determinedfrom a representative number of normal healthy controls or stabletransplant patients.

In another embodiment the percentage of CD4+CD57+ T cells is elevated atleast 2 fold, from 2 to 25 fold, from 2 to 20 fold, from 2 to 15 fold,from 2 to 10 fold, from 2 to 5 fold compared to the percentage ofCD4+CD57+ T cells in a reference.

In another embodiment the CD4+CD57+ T cell content of the blood sampleis measured by flow cytometry. Flow cytometry has been used withincreasing regularity in clinical laboratories for immunophenotyping.The advantages of flow cytometry include speed, sensitivity, precisionand objectivity. The components and operation of flow cytometers is wellknown to those skilled in the art and will not be described in detailherein. For purposes of a description of such, applicants refer to U.S.Pat. No. 5,567,627, issued Oct. 22, 1996 which is incorporated herein byreference in its entirety.

It is sufficient to indicate that the components and methodology of flowcytometry can be used to provide specific information on a number ofparameters of a sample. For example it is possible to provideinformation on components of different sizes within a sample, whilesimultaneously providing information on signals of different wavelengthsreceived from different components received from the sample. Thus, whena sample includes components of varying sizes and also includescomponents with labels which emit different wavelengths of light theflow cytometry data obtained can provide multidimensional information tothe user. The present invention utilizes this technology by providingdifferent types of labeled antibodies and labeled and unlabelled cellsexpressing known antigens. By exposing a sample to such and thereafteranalyzing such within a flow cytometer it is possible to obtainsubstantial amounts of information regarding the blood in a quick andefficient manner.

Multiparameter flow cytometry is the method of choice, whenimmunophenotyping is desired, because more information can beobjectively obtained from fewer cells due to the use of antibodies todifferentiation-associated antigens and antibodies toactivation-associated antigens at the same time to define what functionsspecific populations of cells are performing. For example, multi-colorfluorescence-activated cell sorter analysis can be performed byobtaining peripheral blood samples from patients and normal healthycontrols by hemolysis using the Whole Blood Lysing Reagent Kit (BeckmanCoulter, Fullerton, Calif.). Cell surface antigens of peripheral bloodleukocytes can be detected using a FACS®Calibur flow cytometer with theCELLQuest program (Becton Dickinson, Mountain View, Calif.). Cellsurface antigens and intracellular cytokines can be detected usingfluor-labeled monoclonal antibodies, typically available from BectonDickinson.

Cell stimulation and staining for intracellular cytokine analysis can beperformed according to the manufacturer's instructions (PharMingen, SanDiego, Calif.). Briefly, peripheral blood leukocytes are stimulated withphorbol myristate acetate and calcium ionophore for 4 hours. For thelast 3 hours of stimulation, the intracellular transport inhibitor,nomensin (GOLGISTOP®) is added for accumulation of intracellularcytokines. Afterward, cell surface antigens are stained withfluorescent-labeled monoclonal antibodies specific for the desiredantigen. After CYTOFIX®/CYTOPERM® solution is added to fix andpermeabilize the cells, intracellular cytokines are stained withfluorescent-labeled monoclonal antibodies and multi-colorfluorescence-activated cell sorter (FACS) analysis is performed.

In a further embodiment, the costimulation blockage therapy comprisesadministering L104EA29YIg (belatacept), a mutant CTLA4Ig molecule, whichis a fusion protein that acts as a selective costimulation blocker bybinding surface ligands (CD80 and CD86) on antigen-presenting cells.

The typical belatacept pharmaceutical composition exemplified in theinstant invention is an intravenous (i.v.) formulation listed below.

Composition of Lyophilized L104EA29YIg 100 mg/Vial Drug ProductComponent Amount/Vial (mg)^(a) L104EA29YIg  110^(a) Sucrose 220 SodiumPhosphate Monobasic Monohydrate    15.18 Sodium Chloride    2.55 1NSodium Hydroxide Adjust to pH 7.5 1N Hydrochloric Acid Adjust to pH 7.5^(a)Each vial contains 10% overfill for vial, needle and syringe holdupof the reconstituted solution.

The lyophilized drug is typically reconstituted to about 25 mg/ml with10 ml of either Sterile Water for Injection, USP (SWFI) or 0.9% SodiumChloride Injection, USP. The reconstituted solution is further dilutedto drug product concentrations between 1 and 10 mg/ml with 0.9% SodiumChloride Injection, USP. The diluted drug product for injection isisotonic and suitable for administration by intravenous infusion.

L104EA29Y is typically administered at 10 mg/kg weight of the patientduring the early phase, high risk period that follows transplantationand decreased to 5 mg/kg weight of the patient for a maintenance dosage.

Typical target trough serum concentration of L104EA29Y are between about3 μg/mL and about 30 μg/mL over the first 3 to 6 months post-transplantwhich is sufficient to maintain function of the allograft, preferablybetween about 5 μg/mL and about 20 μg/mL. Typically, target trough serumconcentration of L104EA29Y during the maintenance phase are betweenabout 0.2 μg/mL and about 3 μg/mL, preferably between about 0.25 μg/mLand about 2.5 μg/mL.

A typical administration regimen includes an early phase, in which dosesare higher and the frequency of administration is increased during theperiod of greatest immunologic risk, followed by a maintenance phase.The early phase regimen may range from the first 3 to 6 monthspost-transplantation and involves administration that initially is morefrequent than monthly, preferably as frequently as daily, weekly orevery two weeks depending on the immunologic risk and/or target troughserum concentration. The maintenance phase begins when the early phaseends and involves administration that is not more frequent than monthly,and lasts for as long as needed, typically for as long as the patientretains the transplant. As used herein, day 1 is defined as the day ofthe transplant or the first day of treatment with L104EA29Y orpharmaceutical compositions thereof.

The dosage of L104EA29Y in the early phase is about 8 to about 12 mg/kgweight of the patient, preferably about 10 mg/kg. The dosage ofL104EA29YIg of the invention in the maintenance phase is about 3 toabout 7 mg/kg weight of the patient, preferably about 5 mg/kg.

The early phase may range from the first 3 to 6 monthspost-transplantation. The administration regimen during early phase mayvary depending on the status of the recipient and/or graft. For example,a more intensive early phase regimen would administer a higher dose ofthe molecules or the pharmaceutical compositions of the invention on day1, day 5, week 2 visit (e.g., day 13-17), then every two weeks for thefirst 3 months (e.g., on week 4 visit, week 6 visit, week 8 visit, week10 visit, and week 12 visit), followed by monthly administration throughmonth 6 visit (e.g., on month 4 visit, month 5 visit, and month 6visit). An example of a typical more intensive early phase regimen isadministration of 10 mg/kg weight of the patient of L104EA29YIg at days1, 5, 15, 29, 43, 57, 71, 85, 113, 141 and 169. A less intensiveregimen, for example, would administer the molecules or thepharmaceutical compositions of the invention on day 1, week 2 visit,week 4 visit, then monthly through month 3 visit. An example of atypical less intensive early phase regimen is administration of 10 mg/kgweight of the patient of L104EA29YIg on days 1, 15, 29, 57 and 85.

Typically, an early phase is followed by a maintenance phase where lowerdoses of the molecules or pharmaceutical compositions of the inventionare administered at one to two month intervals for as long as needed,typically for as long as the patient retains the transplant. An exampleof the maintenance phase for the more intensive regimen described aboveincludes monthly administration of 5 mg/kg weight of the patient ofL104EA29YIg starting at month 7 visit. While an example of themaintenance phase for the less intensive regimen above would includemonthly administration of 5 mg/kg weight of the patient of L104EA29YIgstarting at month 4 visit.

Alternatively, one knowledgeable in the art would be able to modify theadministration regimen in response to the patients risk status and/orresponse to the therapy post transplantation. For example, the earlyphase of the less intensive regimen described above could be modified byadding administration day 5 to the regimen, thereby increasing thefrequency of administration during the period of greatest immunologicrisk.

As used herein, “four weeks”, “month”, “months” or “monthly” refers to aperiod of 28±5 days. As used herein, “two weeks” refers to a period of14±3 days.

Flexibility in the administration regimens is required to facilitateadministration scheduling in the lives of transplant recipients, whilemaintaining the target trough profile of L104EA29Y. Permitted windowsfor administering the doses may be as follows:

Visit Visit window Day 1 and day 5 96 hours apart ± 6 hours Week 2Target date ± 2 days Week 4-Month 6 Target date ± 3 days from Month 7thereafter Target date ± 5 days

The target date is a result of adding the desired duration to theprevious actual visit date. The desired duration for the week 2 visit is10 days. The desired duration is 14 days for a visit planned for twoweeks from the previous visit, e.g., a week 6 visit following a week 4visit. The desired duration is 28 days for a visit planned for a monthor four week from the previous visit, e.g., a month 4 visit following amonth 3 visit. The desired duration is 56 days for a visit planned fortwo months from the previous visit, e.g., a month 8 visit after a month6 visit. For example, a day 15 actual visit date plus 14 days results ina week 4 target date of day 29. Based on the visit windows above, theadministration may occur on day 29±3 days. Should the administrationoccur on day 26, that day becomes the actual visit date utilized for thecalculation of the next target date.

Low risk of acute rejection recipients typically include those whoreceive transplants from living related donors and well matchedrecipient/donors. High risk of acute rejection recipients typicallyinclude those who receive transplants from marginal donors orre-transplants, have high panel reactive antibodies or are AfricanAmerican.

As will be apparent to those skilled in the art to which the inventionpertains, the present invention may be embodied in forms other thanthose specifically disclosed above without departing from the spirit oressential characteristics of the invention. The particular embodimentsof the invention described above, are, therefore, to be considered asillustrative and not restrictive. The scope of the present invention isas set forth in the appended claims rather than being limited to theexamples contained in the foregoing description.

EXAMPLE 1

Early costimulation blockade resistant rejection has been seen in somepatients on belatacept-based immunosuppression. Hypothesizing that Tcell phenotype might prospectively identify patients at risk forcostimulation blockade resistant rejection, patients receivingbelatacept-based costimulation blockade, were studied by specificallyquantifying terminally differentiated memory cells expressing CD57, amarker of T cell senescence, and relating this expression to clinicaloutcome.

Methods: Samples from renal allograft recipients receivingbelatacept-based immunosuppression were selected from an IRB-approvedtissue acquisition protocol.

Study Population:

Organ transplant recipients, candidates for organ transplantation, andtheir organ donors under evaluation at the Emory UniversityHospital/Emory Transplant Center (Emory) or Children's Healthcare ofAtlanta (CHOA) were considered for study.

Inclusion Criteria:

-   -   Recipients of or candidates for organ transplantation or organ        donors for recipients under evaluation at Emory or CHOA.    -   Normal volunteers to include individuals without any known        end-stage organ disease who are not on any immunosuppressive        medication, and individuals with conditions requiring        immunosuppression (such as dermatological diseases) that do not        require transplant therapies.        Exclusion Criteria:    -   Patients who fail to meet the criteria for transplantation or        post transplant follow-up by Emory or CHOA physicians.    -   Any condition that, in the opinion of the attending physician,        would place the patient at undue risk by participating. Specific        conditions include but are not limited to anemia prohibitive of        phlebotomy, coagulopathy or technical considerations that would        prevent acquisition of sufficient tissue on biopsy for clinical        use, or medical urgency preventing timely administration of the        consenting process.        Enrollment

Volunteers were recruited for each of the following scenarios:

-   -   Group 1: Development of a clinically recognized complication        with potential immune etiology or ramifications. Examples        include opportunistic infection, rejection, malignancy,        alloantibody formation, or immunosuppressive drug toxicity.    -   Group 2: Maintenance of immune stability characterized by stable        graft function without evident complication. These patients        served as comparators for individuals with specific        complications under scenario 1. These individuals were enrolled        to establish demographically matched cohorts of up to 20        individuals for each identified complication identified in Group        1.    -   Group 3: Pre-transplant status. Recipient candidates for kidney,        pancreas, liver or lung transplantation were enrolled and they        were followed through the waiting period, transplantation and        post transplantation. These individuals served to prospectively        capture immune complications prior to clinical presentation of        an immune complication to facilitate the retrospective        evaluation of assays with potential utility in the prospective,        sub-clinical detection of burgeoning complications.    -   Group 4: Donors for individuals meeting the criteria for one of        the 3 above mentioned scenarios. As many of the immune related        complications are triggered by or involve immune responses that        are specific for donor antigens, donor tissue ease required for        the assessment of donor specific immunity. As kidney transplant        recipients received organs from live donors, their donors were        enrolled to provide target peripheral blood lymphocytes for        mechanistic analysis.    -   Group 5: Individuals with liver, renal, or pulmonary diseases        that may lead to the development of organ failure. Including        these individuals allowed the study of mechanisms of diseases        that may recur post transplantation, and better enable one to        predict which patients are at increased risk for recurrence.        Moreover, this group allowed the study of disease complications        that may affect the post transplantation course.    -   Group 6: Normal volunteers and people on immunosuppressive        agents for non-transplanted conditions were recruited to serve        as reference comparators for patient samples.

Patients were recruited in a manner that was sensitive to the inclusionof women and members of underserved minority groups. Enrollment targeteddemographics that generated a collection of samples from individuals ofage, gender, and ethnicity as appropriate for the disease process underevaluation.

Procedure

Following enrollment volunteers were able to donate samples. In general,the volume of blood donated for this study was 50 mL or 3 mL/kg,whichever was less, for each time point. Under circumstances where theneeds of a specific assay require larger amounts (e.g., in lymphocytedepleted patients, or multiple planned assays), a larger blood volume,up to a maximum of 150 mL for any single phlebotomy session wascollected and did not exceed 3 mL/kg. The total amount of blood drawndid not exceed 450 mL, or 7 mL/kg whichever was less, over any 6-weekperiod in keeping with current NIH guidelines. Biopsy tissue and BALfluid was limited to tissue or fluid in excess of that required forclinical diagnostic needs, as determined by the physician overseeing theprocedure. Urine was limited to that which can be donated withoutinterfering with diagnostic testing, and was typically only collectedfrom normal volunteers and kidney donors or recipients. The donationschedule varied by group. In general, patients with documentedcomplications (Group 1) were enrolled and studied at the time thecomplication was diagnosed, 1 and 6 months following the resolution (orpersistence in the case of chronic conditions) of the complication.

Samples were delivered to the Emory Transplant Biorepository where itwas processed for study or storage. In general, blood samples were beseparated and stored in the form of peripheral blood mononuclear cells(PBMC), serum, plasma, PBMC derived DNA, and PBMC derived RNA convertedto cDNA. Biopsy tissue was snap frozen in liquid nitrogen and convertedto DNA, RNA, and cDNA. BAL fluid was separated into a cellular fluid andmononuclear cells, and cells were stored or converted to DNA, RNA, andcDNA. Urine was spun and separated in to a cellular supernatants andcell pellets. Pellets were stored and/or converted to RNA and cDNA.Samples may have been studied fresh or discarded as excess.

Demographic data including age, gender, and race was recorded to assurediversity of this population. Clinical information was collected at thetime of each sample collection and included information such as primarydisease requiring transplant, date of transplant, date of sampledonation, medication dose at time of transplant, most recent level ofimmunosuppressants, creatinine at time of donation, and baselinecreatinine. Each time a subject was asked to donate blood their healthstatus will be reviewed. Clinically concerning information that becomesapparent as a result of study visits (e.g., excessive bruising postphlebotomy, gross hematuria, etc.) was relayed to the patient'sattending physician for disposition.

Assay

Peripheral blood samples obtained prior to drug administration andanalyzed by multiparameter flow cytometry, specifically analyzing thesamples for markers of memory, differentiation, activation, exhaustionand senescence.

The PBMC samples were thawed and resuspended at 5×10⁵ cells/SOW/tube. 1μl live/dead fixable aqua dead cell stain was added per tube. The tubeswere then incubated in the dark at room temperature for 30 mins and thenwashed 2 times with 1 ml 1×PBS and resuspended in 50 μl of PBS.

Antibodies: The antibodies conjugated to fluorescein isothiocyanate(FITC) included CD2 (BD Pharmingen #555326), CD11a (BioLegend #301206),CD39 (Miltenyi Biotec #130-093-502), CD57 (BD Pharmingen #555619), CD103(Beckman Coulter #IM1856U), IgD (BD Pharmingen #555778), Ki67 (BDPharmingen #556026), and Perforin (BD Pharmingen #556577).

The antibodies conjugated to phycoerythrin (PE) included CD25 (MiltenyiBiotec #130-091-024), CD28 (BD Pharmingen #555729), CD31 (MiltenyiBiotec #130-092-653), CD38 (BD Pharmingen #555460), BCL2 (BD Pharmingen#556535), IgM (BD Pharmingen #555783), PD1 (CD279) (BioLegend #329906)and Granzyme B (Invitrogen #MHGB04).

PE-Cy7 is a tandem fluorochrome composed of R-phycoerythrin (PE), whichis excited by 488-nm light and serves as an energy donor, coupled to thecyanine dye Cy7(Cy7), which acts as an energy acceptor and fluorescesmaximally at 780 nm. The antibodies conjugated to PE-Cy7 tandemfluorochrome included CD19 (BD Pharmingen #341093), CD127 (eBioscience#25-1278-42), and CCR7(CD197) (BD Pharmingen #557648).

The antibodies conjugated to allophycocyanin (APC) included CD27(BioLegend #302810), HLADR (BD Pharmingen #340549), CCR5(CD195) (R&D#FAB1802A), and IgG (BD Pharmingen #550931). CD127 (BD Pharmingen#558598) was conjugated to ALEXA FLUOR®647. ALEXA FLUOR®647 fluorochromeemission is collected at the same instrument settings as for APC.

The antibodies conjugated to ALEXA FLUOR®700 included CD3 (BD Pharmingen#557943), and CD27 (eBioscience #50-168-83).

The antibodies conjugated to APC-eFluor 780 CD8 (eBioscience#47-0088-42) and CD24 (eBioscience #47-0247-42).

The antibodies conjugated to V450 included CD4 (BD Horizon #560345) andCD5 (BD Horizon #644487).

The antibodies conjugated to Pacific Orange (PacOrange) included CD3(Invitrogen #CD0330TR), CD14 (Invitrogen #MHCD1430) and CD20 (Invitrogen#MHCD2030).

The antibodies conjugated to QDOT®655 nanocrystal conjugate (QDOT®655)included CD45RA (Invitrogen #Q10069). CD38 (eBioscience #95-0388-42) wasconjugated to eFluor650NC. eFluor650NC fluorochrome emission iscollected at the same instrument settings as for QDOT®655.

The Extracellular Stain: The required amount of antibodies listed inFIG. 4 were added to the appropriate tubes and incubated in the dark onice for 30 mins, then washed 2 times with 1 ml 1×PBS and resuspended in200 μl of PBS.

The Intracellular Stain: 300 μl BD CYTOFIX®/CYTOPERM® was added to tubes7and 8 and incubated in the dark, on ice for 30 mins and then washed 2times with 1 ml 1×BD Perm/Wash buffer. ICS antibodies shown in FIG. 4were added to the appropriate tubes and incubated in the dark at roomtemperature for 30 mins, then washed 2 times with 1 ml 1×BD Perm/Washbuffer and resuspended in 200 μl of PBS.

The samples were run on a BD LSRFortessa (BD Biosciences, San Jose,Calif.) according to the manufactures instructions.

PCR analysis of telomere length, a parameter relevant to senescence,also was performed with QuantStudio (Life Technologies) with iTaq SYBR®(Bio-Rad) and compared to reference samples from 5 normal healthycontrols. Within 7 months of transplantation, 9 patients experiencedacute cellular rejection and 5 were rejection-free.

Results: Patients who eventually experienced acute cellular rejectionhad a markedly higher percentage of CD4+CD57+PD1− T cells (p=0.0055) atthe time of transplantation compared to patients without rejection (FIG.1). Although typically considered a marker of senescence, CD57expression on CD4+ T cells was paradoxically correlated with expressionof the proliferation marker Ki67 (p=0.0087), and was not associated withshorter telomere length (p=0.7424). These relationships were limited tothe CD4 compartment. CD8+CD57+ T cells marginally trended higher inacute cellular rejection patients (p=0.2358, FIG. 1), but CD57expression on CD8+ T cells was associated with more traditional indicesof senescence including reduced Ki67 expression and shorter telomerelength.

Discussion: This study identified a CD4+ T cell phenotype that stronglycorrelates with risk for belatacept-resistant rejection. CD57 has beencommonly described as an external marker of cell senescence, a state inwhich the cell is in persistent cell cycle arrest. However, thenon-senescent (Ki67+, normal telomere length) phenotype associated withCD57, and its limitation to CD4+ T cells suggests that in this settingit is a FLAG® for activated T follicular helper (TFH) cells. TFH cellsare antigen-experienced cells known to be critical for certain immuneresponses and driven by alternative costimulatory pathways includingCD154 and the CD28 homologue ICOS.

What is claimed is:
 1. A method of treating a transplant patient at riskof costimulation blockade therapy rejection, the method comprising: (a)providing a transplant patient or a patient in need of a transplanthaving at least a 2-fold increase in CD4+CD57+T cells as compared to anormal healthy control; and (b) administering belatacept to thetransplant patient wherein belatacept is administered to the transplantpatient pursuant to an early phase regimen and a maintenance phaseregimen; wherein the early phase regimen comprises administering 10mg/kg of belatacept to the transplant patient during the first three tosix months post-transplantation; and wherein the maintenance phaseregimen comprises administering 5 mg/kg of belatacept monthly to thetransplant patient following the early phase regimen and continuing forthe life of the transplant.
 2. The method according to claim 1, whereinthe early phase regimen comprises administering belatacept on the day ofthe transplant, about 96 hours post-transplant, two weekspost-transplant, four weeks post-transplant, eight weeks post-transplantand twelve weeks post-transplant.
 3. The method according to claim 2,further comprising administering belatacept sixteen weekspost-transplant, twenty weeks post-transplant and twenty-four weekspost-transplant.
 4. The method according to claim 1, wherein thetransplant comprises solid organ, tissue and/or cell.
 5. The methodaccording to claim 4, wherein the transplant comprises skin, isletcells, muscles, hepatocytes, neurons, heart, liver, kidney, lung,appendages, limbs, nose, ear or face.
 6. The method according to claim4, wherein the transplant is a kidney.